By John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin
This quantity emphasizes equipment for the assay, purification, and characterization of adenylyl cyclases, guanine nucleotide-dependent regulatory proteins (G proteins), and guanylyl cyclases.
* Adenylyl cyclase: decision of activity
* education of fabrics beneficial in purification of parts of hormonally responsive cyclase systems
* Purification guanine nucleotide-dependent regulatory proteins
* Purification and characterization of g proteins: labeling and quantitating of G proteins
* Reconstitution guanylyl cyclase: assay
* practise of fabrics used for examine of the cyclase
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Additional info for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
2 mM cAMP. Continue to next paragraph. 25 ml incubation medium. Keep test tubes cold for 30 min, with occasional shaking, and centrifuge to obtain a clear, protein-free PCA supernatant containing the extracted 3H-labeled nucleotides. 9 ml (MX) of the clear PCA supernatant to a set of marked test tubes and prepare extracts for chromatography (see below). Procedure for Tissue Slices It is recommended that the experiment be performed in triplicate. t The use of trypsin, at this stage, should be avoided due to potential damage to receptors and peptide hormones.
Put all the labeled slices on a sheet of Parafilm placed on an insulating layer of tissue paper and rapidly divide the slices into equal portions (25-100 mg/experimental point) with a spatula (make sure that the tissue does not dry by keeping sufficient medium for wetting on the Parafilm sheet). 5-1 rat lacrimal gland, as used in the author's laboratory) represents one experimental point. To quickly achieve easy division, divide the total material into two equal portions and each half in two equal portions, etc.
Biochem. 85, 451 (1978). 20 ADENYLYLCYCLASE  For this reason sample recovery is determined either with unlabeled cAMP or with cAMP that is labeled with a second isotope. In addition, since many adenylyl cyclases exhibit low activities, especially under basal assay conditions, the radioactivity measured in the sample in the absence of enzyme (no enzyme blank) can represent sizable percentage of that measured with enzyme. Consequently, it becomes important to consider how this value is to be treated in the calculation of activity.